CN104726430B - The α galactosidase As gaAHJ8 and its gene of salt tolerant resistant protease - Google Patents

Abstract

The invention discloses a salt-resistant and protease-resistant α-galactosidase AgaAHJ8 and its gene. The present invention provides an α-galactosidase AgaAHJ8 whose amino acid sequence is shown in SEQ ID NO.1, the gene agaAHJ8 encoding the above-mentioned α-galactosidase, a recombinant vector containing the above-mentioned gene, and a recombinant strain containing the above-mentioned gene . The α-galactosidase of the present invention maintains more than 72% of the enzyme activity in the range of pH 4.0-8.0; the optimum temperature is 50°C, and is stable at 37°C; it has good salt tolerance and protease resistance, Hydrolyzable disaggregates, raffinose and guar gum. The α-galactosidase AgaAHJ8 of the present invention can be applied in the field of high-salt food and seafood processing, especially in the fermentation of high-salt food (such as high-salt dilute soy sauce).

Description

Salt-tolerant protease-resistant α-galactosidase AgaAHJ8 and its gene

technical field

The invention relates to the technical field of genetic engineering, in particular to a salt-resistant and protease-resistant α-galactosidase AgaAHJ8 and its gene.

Background technique

Oligosaccharides and polysaccharides with α-galactoside are widely found in food and feed materials, especially legume seeds have the highest content (Karr-Lilienthal et al., Livest Prod Sci, 2005, 97:1– 12.), such as oligosaccharides such as melibiose, raffinose, stachyose and verbascose, and galactomannans such as carob gum and guar gum. α-galactosidase (α-galactosidase, EC 3.2.1.22), also known as melibiase, can catalyze the hydrolysis of α-galactosidic bonds in these oligosaccharide and polysaccharide substrates, and then apply In feed, food, paper and medical industries (Zhou et al., Appl Microbiol Biotechnol, 2010, 88:1297–1309).

Salt-tolerant enzymes still have catalytic activity under high concentrations of NaCl, and can be used in high-salt food and seafood processing and other high-salt environment biotechnology fields. Processing food in high-salt environments can also prevent microbial contamination and save sterilization, etc. energy consumed (Zhou et al.Folia Microbiol,2014,59:423–431); since endogenous proteases are widely present in organisms and exogenous proteases are often used as an additive, protease-resistant enzymes can be used in food and other industries (Zhou et al.Folia Microbiol, 2014,59:423–431). For example, the soy sauce produced by the high-salt dilute state process has high quality and good flavor. The concentration of sodium chloride in the soy sauce or moromi reaches 18% (w/v). The raw materials for making soy sauce contain endogenous protease, which can be added during fermentation Exogenous protease and galactosidase, but because the enzyme activity is inhibited under the condition of high concentration of sodium chloride, the fermentation of high-salt dilute soy sauce has long fermentation cycle, low equipment utilization rate, raw material utilization rate and amino nitrogen production Low efficiency and other shortcomings (patent: 201110248389.8).

Contents of the invention

The object of the present invention is to provide a salt-resistant and protease-resistant α-galactosidase AgaAHJ8.

Another object of the present invention is to provide a gene encoding the above-mentioned α-galactosidase.

Another object of the present invention is to provide a recombinant vector comprising the above gene.

Another object of the present invention is to provide recombinant strains containing the above genes.

The α-galactosidase AgaAHJ8 of the present invention can be obtained from Pontibacter sp. The amino acid sequence of AgaAHJ8 is shown in SEQ ID NO.1.

The α-galactosidase AgaAHJ8 of the present invention contains a total of 402 amino acids, with a theoretical molecular weight of 45.2kDa, wherein the N-terminal 19 amino acids are the predicted signal peptide sequence "MKKLFLFFFLNLYIGAAYA", and the mature α-galactosidase AgaAHJ8 contains 383 amino acids . The optimum pH value of the enzyme is 5.5, and more than 72% of the enzyme activity can be maintained in the range of pH 4.0-8.0; after being treated with pH 4.0-8.0 buffer at 37°C for 1 hour, the enzyme activity remains up to 73% above. The optimal temperature of the enzyme is 50°C, it is stable at 37°C, and its half-life is less than 5 minutes at 50°C. At pH 5.5 and 50°C, the specific activity of the enzyme to 2mM pNPG (p-nitrophenyl-α-D-galactopyranoside) is 43.5±1.0Umg ^-1 , and to 0.5% (w/v) melibiose ), raffinose and guar gum were 0.47±0.07, 1.93±0.07 and 0.46±0.06U mg ^-1 , respectively. The enzyme has good salt tolerance, and when 3.5-30.0% (w/v) NaCl is added to the reaction system, it still has more than 70% enzyme activity. The enzyme also has good protease resistance, and after being treated with trypsin and proteinase K at 37°C for 1 hour, the enzyme can still maintain 101.1% and 108.9% of the enzyme activity, respectively.

The present invention provides the gene agaAHJ8 encoding the above-mentioned α-galactosidase, and the gene sequence is shown in SEQ ID NO.2.

The present invention clones the coding gene agaAHJ8 of α-galactosidase AgaAHJ8 by genome sequencing method, its full length is 1209bp, the start codon is ATG, and the stop codon is TAG. By BLAST comparison, the complete sequence of the α-galactosidase AgaAHJ8 has the highest amino acid sequence identity of 69.2% with the complete sequence of the hypothetical protein (hypothetical protein) N824_24065 (ETZ22001) derived from Pedobacter sp.V48 in GenBank, which is 69.2%. The activity of the protein derived from sp.V48 has not been studied yet; the identity of the complete sequence of AgaAHJ8 with the complete sequence of α-galactosidase (B3PGJ1) derived from Cellvibrio japonicus Ueda107 whose activity was confirmed is 44.5%. It shows that α-galactosidase AgaAHJ8 is a new α-galactosidase.

The present invention also provides a recombinant vector comprising the above-mentioned α-galactosidase gene agaAHJ8, preferably pEasy-E2-agaAHJ8. The α-galactosidase gene of the present invention is inserted into the expression vector, and its nucleotide sequence is connected with the expression control sequence. As a most preferred embodiment of the present invention, the α-galactosidase gene of the present invention and the expression vector pEasy-E2 are connected by T-A method to obtain the recombinant Escherichia coli expression plasmid pEasy-E2-agaAHJ8.

The present invention also provides a recombinant strain comprising the above-mentioned α-galactosidase gene agaAHJ8, preferably the strain is Escherichia coli, yeast, Bacillus or Lactobacillus, preferably the recombinant strain BL21(DE3)/agaAHJ8.

The method for preparing α-galactosidase AgaAHJ8 of the present invention is carried out according to the following steps:

1) Transforming host cells with the above-mentioned recombinant vectors to obtain recombinant strains;

2) Cultivate the recombinant strain to induce the expression of recombinant α-galactosidase;

3) recovering and purifying the expressed α-galactosidase AgaAHJ8.

Wherein, the host cell is preferably an E. coli cell, and the recombinant E. coli expression plasmid is preferably transformed into an E. coli cell BL21(DE3) to obtain a recombinant strain BL21(DE3)/agaAHJ8.

The present invention provides a new α-galactosidase gene, the α-galactosidase coded by it has an optimal pH of 5.5, an optimal temperature of 50°C, and can hydrolyze disaccharides, raffinose and guar gum, Has good salt and protease resistance properties. The enzyme can be applied in the fields of high-salt food and seafood processing.

Description of drawings

Figure 1: SDS-PAGE analysis of recombinant α-galactosidase AgaAHJ8 expressed in Escherichia coli, wherein, M: protein marker; 1: purified recombinant α-galactosidase AgaAHJ8;

Figure 2: pH activity of purified recombinant α-galactosidase AgaAHJ8;

Figure 3: pH stability of purified recombinant α-galactosidase AgaAHJ8;

Figure 4: Thermal activity of purified recombinant α-galactosidase AgaAHJ8;

Figure 5: Thermal stability of purified recombinant α-galactosidase AgaAHJ8;

Figure 6: Activity of purified recombinant α-galactosidase AgaAHJ8 in different concentrations of NaCl.

detailed description

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Test materials and reagents

1. Bacterial strains and vectors: Pontibacter sp. is the same as that reported in the literature, such as Pontibacter sp. CICC 23788 from the China Industrial Microbiology Culture Collection Management Center; Escherichia coli BL21 (DE3) and expression vector pEasy-E2 were purchased From Beijing Quanshijin Biotechnology Co., Ltd.

2. Enzymes and other biochemical reagents: DNA polymerase and dNTP were purchased from TaKaRa; pNP (p-nitrophenol), pNPG (p-nitrophenyl-α-D-galactopyranoside), galactose and melibiose were purchased from Sigma Company; raffinose (raffinose) was purchased from Amresco Company of the United States; others were all domestic reagents (all of which could be purchased from common biochemical reagent companies).

3. Medium:

LB medium: Peptone 10g, Yeast extract 5g, NaCl 10g, add distilled water to 1000ml, pH natural (about 7). Add 2.0% (w/v) agar to the solid medium on this basis.

Explanation: For the molecular biology experimental methods not specifically described in the following examples, all refer to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or follow the kit and product manual.

Example 1: Cloning of α-galactosidase gene agaAHJ8

Extracting the genomic DNA of marine bacilli: centrifuge the bacterial liquid cultured in liquid for 2 days to take the bacterial cells, add 1mL lysozyme, treat at 37°C for 60min, and then add the lysate, the lysate consists of: 50mM Tris, 20mM EDTA, 500mM NaCl, 2% ( w/v) SDS, pH 8.0, lysed in a water bath at 70°C for 60 minutes, mixed every 10 minutes, and centrifuged at 10,000 rpm for 5 minutes at 4°C. The supernatant was extracted in phenol/chloroform to remove impurity proteins, and then an equal volume of isopropanol was added to the supernatant. After standing at room temperature for 5 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuum, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

5 μg of the marine bacilli genome was broken into 400–600 bp fragments with an ultrasonic breaker Biorupter, and the broken DNA fragments were purified with the Genomic DNA Clean&Concentration kit, and the DNA fragments were complemented with the TureseqTM DNA Sample Preparation Kit after purification Add A bases and linkers to the blunt, 3' ends, and PCR amplification of DNA fragments (operate according to the instructions of the kit). Genome sequencing was performed on the above-prepared library using a MiSeq genome sequencer (Illumima).

The data obtained by genome sequencing were predicted by reading frame and compared with local BLAST, and the α-galactosidase gene agaAHJ8 was obtained, and the gene sequence is shown in SEQ ID NO.2.

Embodiment 2: Preparation of recombinant α-galactosidase AgaAHJ8

5'CAACAGAAGGCATCCCTTGCCCCC 3' and 5'GATCTTTTTGAGGCGGAAAAGCTTTG 3' were used as primer pairs and the genomic DNA of Marine bacteria was used as template for PCR amplification. The PCR reaction parameters were: denaturation at 94°C for 5 min; then denaturation at 94°C for 30 sec, annealing at 65°C for 30 sec, extension at 72°C for 2 min and 30 sec, and after 30 cycles, incubation at 72°C for 10 min. As a result of PCR, the α-galactosidase gene agaAHJ8 was obtained, and a protruding A base was introduced at the 3' end of the gene. The α-galactosidase gene agaAHJ8 and the expression vector pEasy-E2 were connected by T-A method to obtain the recombinant expression plasmid pEasy-E2-agaAHJ8 containing agaAHJ8. Transform pEasy-E2-agaAHJ8 into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli strain BL21(DE3)/agaAHJ8.

The recombinant Escherichia coli strain BL21(DE3)/agaAHJ8 containing the recombinant plasmid pEasy-E2-agaAHJ8 was inoculated in LB (containing 100 μg mL ^-1 Amp) culture medium at an inoculum size of 0.1%, and shaken rapidly at 37°C for 16 hours. Then inoculate the activated bacterial solution into fresh LB (containing 100 μg mL ^-1 Amp) culture solution with 1% inoculum, and after rapid shaking culture for about 2–3 hours (OD ₆₀₀ reaches 0.6–1.0), add a final concentration of 0.1 Induced by mM IPTG, continue shaking culture at 20°C for about 20h or 26°C for about 8h. Centrifuge at 12000rpm for 5min to collect the bacteria. After suspending the cells with an appropriate amount of pH 7.0 Tris–HCl buffer, the cells were disrupted by ultrasonic waves in a low-temperature water bath. The crude enzyme solution concentrated in the cells above was centrifuged at 13,000rpm for 10min, the supernatant was aspirated and the target protein was affinity and purified with Nickel-NTA Agarose and 0-500mM imidazole respectively. The results of SDS-PAGE (Fig. 1) showed that the recombinant α-galactosidase AgaAHJ8 was expressed in Escherichia coli, and the product was a single band after purification.

Example 3: Determination of the properties of the purified recombinant α-galactosidase AgaAHJ8

1. Activity analysis of purified recombinant α-galactosidase AgaAHJ8

The activity determination method of the purified recombinant α-galactosidase AgaAHJ8 adopts the pNPG method: dissolve pNPG in 0.1M buffer solution to make the final concentration 2mM; After preheating at the reaction temperature for 5 minutes, add the enzyme solution and react for 10 minutes, then add 2 mL of 1M Na ₂ CO ₃ to terminate the reaction, and measure the released pNP at a wavelength of 405 nm after cooling to room temperature; 1 enzyme activity unit (U) Defined as the amount of enzyme required to decompose pNPG to produce 1 μmol pNP per minute. The method for measuring the activity of the substrate raffinose and guar gum adopts the DNS method: the substrate is dissolved in 0.1M buffer solution to make the final concentration 0.5%; the reaction system contains 50 μL of an appropriate amount of enzyme solution, 450 μL of the substrate; After preheating at the reaction temperature for 5 minutes, add the enzyme solution and react for an appropriate time, then add 2.0mL DNS to terminate the reaction, cook in boiling water for 5 minutes, and measure the OD value at a wavelength of 540nm after cooling to room temperature; 1 enzyme activity unit (U) It is defined as the amount of enzyme required to decompose the substrate to produce 1 μmol reducing sugar (calculated as galactose) per minute under the given conditions. The method for measuring the activity of the substrate melibiose adopts the glucose oxidase method: dissolve the substrate in 0.1M buffer to make the final concentration 0.5% (w/v); After the substrate was preheated at the reaction temperature for 5 minutes, the enzyme solution was added to react for 10 minutes, and then according to the principle of glucose oxidase-peroxidase method, the glucose determination kit (Shanghai Rongsheng Bio-Pharmaceutical Co., Ltd., CAT361500 ) Instructions for measuring enzyme activity; 1 enzyme activity unit (U) is defined as the amount of enzyme required to decompose the substrate to produce 1 μmol of glucose per minute under given conditions.

2. Determination of pH activity and pH stability of purified recombinant α-galactosidase AgaAHJ8:

Optimum pH determination of the enzyme: α-galactosidase AgaAHJ8 was subjected to an enzymatic reaction at 37°C in 0.1M pH 3.0–10.0 buffer. Determination of the pH stability of the enzyme: the enzyme solution was placed in a 0.1M pH3.0–10.0 buffer, treated at 37°C for 1 hour, and then the enzymatic reaction was carried out at pH 5.5 and 37°C, and the untreated enzyme liquid as a control. The buffer solution is: 0.1MMcIlvaine buffer (pH3.0-8.0) and 0.1M glycine-NaOH (pH9.0-10.0). Using pNPG as a substrate, reacted for 10 minutes, and measured the enzymatic properties of the purified AgaAHJ8. The results showed that the optimum pH of AgaAHJ8 was 5.5, and more than 72% of the enzyme activity could be maintained in the range of pH 4.0–8.0 (Figure 2); Survival of more than 73% (Figure 3).

3. Determination of thermal activity and thermal stability of purified recombinant α-galactosidase AgaAHJ8:

Enzyme thermal activity assay: The enzymatic reaction was carried out at 10–70°C in pH 5.5 buffer. Determination of enzyme thermal stability: put the same amount of enzyme solution at 37°C and 50°C respectively, and after treatment for 0-60 minutes, carry out enzymatic reaction at pH 5.5 and 37°C, and use untreated enzyme solution as a control . Using pNPG as a substrate, reacted for 10 min, and measured the enzymatic properties of the purified AgaAHJ8. The results showed that the optimal temperature of AgaAHJ8 was 50°C (Figure 4); the enzyme was stable at 37°C, and the half-life was less than 5min at 50°C (Figure 5).

4. Determination of kinetic parameters of purified recombinant α-galactosidase AgaAHJ8:

Determination of enzyme kinetic parameters First-order reaction time: at pH 5.5 and 50°C, with 1mM pNPG or 14mM melibiose as the substrate, the reaction is terminated within 1-30min of the enzymatic reaction and the enzyme activity is measured. Calculate the ratio of enzyme activity to reaction time. If the ratio remains stable within a certain period of time, this time is the first-order reaction time. Using 0.05–2.0mM pNPG or 2.9–36.5mM melibiose as the substrate, at pH 5.5, 50°C and first order reaction time, K _m , V _max and k _cat were determined according to the Lineweaver–Burk method. It was determined that the K _m , V _max and k _cat of AgaAHJ8 to pNPG were 2.0 mM ^-1 , 111.1 μmol min ^-1 mg ^-1 and 83.2 s ^-1 at 50°C and pH 5.5, respectively. The K _m , V _max and k _cat are 13.2mM ^-1 , 0.9μmolmin ^-1 mg ^-1 and 0.7s ^-1 , respectively.

5. Effects of different metal ions and chemical reagents on the activity of purified recombinant AgaAHJ8:

Add 1.0mM metal ions and chemical reagents to the enzymatic reaction system to study their influence on the enzyme activity. Under the conditions of 37°C and pH5.5, the enzyme activity was measured with pNPG as substrate. The results (Table 1) showed that 1.0mM SDS, HgCl ₂ and AgNO ₃ could completely or almost completely inhibit AgaAHJ8; other metal ions and chemical reagents had little effect on AgaAHJ8.

Table 1. Effects of metal ions and chemical reagents on the activity of purified recombinant α-galactosidase AgaAHJ8

6. Activity of purified recombinant α-galactosidase AgaAHJ8 in NaCl:

Determination of enzyme activity in NaCl: add 3.5-30.0% (w/v) NaCl to the enzymatic reaction system, and carry out the enzymatic reaction at pH 5.5 and 37°C. Using pNPG as a substrate, reacted for 10 min, and measured the enzymatic properties of the purified AgaAHJ8. The results show that: AgaAHJ8 has good salt tolerance, and the enzyme still has more than 70% of the enzyme activity when 3.5-30.0% (w/v) NaCl is added to the reaction system (Figure 6).

7. Determination of protease resistance of purified recombinant α-galactosidase AgaAHJ8:

Protease resistance of the enzyme: Treat the recombinant enzyme with trypsin (pH7.5) and proteinase K (pH7.5) equivalent to 45 times (w/w) of the recombinant enzyme at 37 ° C for 1 h, then at pH 5.5 and 37 The enzymatic reaction was carried out at ℃, and the enzyme solution placed in the pH buffer corresponding to the protease but without protease was used as a control. The results showed that AgaAHJ8 could retain 101.1% and 108.9% of the enzyme activity after treatment with trypsin and proteinase K at 37°C for 1 hour, respectively.

8. Degradation of substrate by purified recombinant α-galactosidase AgaAHJ8:

At pH 5.5 and 50°C, the specific activity of the enzyme to 2mM pNPG is 43.5±1.0U mg ^-1 , and to 0.5% (w/v) melibiose, raffinose and guar The specific activities of guar gum were 0.47±0.07, 1.93±0.07 and 0.46±0.06 U mg ^-1 , respectively.

The above embodiments are intended to illustrate that the present invention can be implemented or used by those skilled in the art. It will be obvious to those skilled in the art to modify the above embodiments, so the present invention includes but is not limited to the above embodiments. Any method, process, or product that conforms to the claims or the description of the specification, and conforms to the principles, novelty, and creative features disclosed herein falls within the protection scope of the present invention.

Claims (4)

1. a kind of alpha-galactosidase A gaAHJ8 of salt tolerant resistant protease gene, it is characterised in that with 5' CAACAGAAGGCATCCCTTGCCCCC 3' and 5'GATCTTTTTGAGGCGGAAAAGCTTTG 3' are primer pair, ocean bacillus Genomic DNA is template, enters performing PCR amplification；PCR response parameters are：94 DEG C of denaturation 5min；Then 94 DEG C denaturation 30sec, 65 DEG C Anneal 30sec, 72 DEG C of extension 2min 30sec, 72 DEG C of insulation 10min after 30 circulations；

The ocean bacillus is Pontibactersp., is Chinese industrial Microbiological Culture Collection administrative center bacterial strain Pontibactersp.CICC 23788。

2. include the recombinant vector of encoding gene described in claim 1.

3. the host cell containing recombinant vector described in claim 2.

4. host cell according to claim 3, it is characterised in that the host cell is Escherichia coli, saccharomycete, bud Spore bacillus or Bacillus acidi lactici.